Albumin is a highly soluble, ellipsoidal protein (MW 66,500), accounting for 70–80% of the colloid osmotic pressure of plasma. Accordingly, albumin is important in regulating the volume of circulating blood. When injected intravenously, 5% albumin will increase the circulating plasma volume by an amount approximately equal to the volume infused. This extra fluid reduces hemoconcentration and decreases blood viscosity. The degree and duration of volume expansion depend upon the initial blood volume. When treating patients with diminished blood volume, the effect of infused albumin may persist for many hours. In individuals with normal blood volumes, the hemodilution lasts for a much shorter time.
Albumin is also a transport protein and binds naturally occurring, therapeutic, and toxic materials in the circulation.
Albumin is distributed throughout the extracellular water and more than 60% of the body albumin pool is located in the extravascular fluid compartment. The total body albumin in a 70 kg man is approximately 350 g; it has a circulating life span of 15–20 days, with a turnover of approximately 15 g per day.
The minimum serum albumin level necessary to prevent or reverse peripheral edema is unknown. Although it undoubtedly varies from patient to patient, there is some evidence that it falls near 2.5 g per deciliter. This concentration provides a plasma oncotic pressure of 20 mm Hg (the equivalent of a total protein concentration of 5.2 g/dL).
Preparations containing albumin, including plasma protein fractions, are often provided therapeutically to humans and animals. For example, preparations containing albumin are frequently administered to humans for one or more of the following indications: hypovolemia, with or without shock; hypoalbumenimia, which may result from inadequate production of albumin (due to malnutrition, burns, major injury, congenital analbuminemia, liver disease, infection, malignancy, or endocrine disorders), excessive catabolism (due to burns, major injury, pancreatitis, thyrotoxicosis, pemphigus, or nephrosis), loss of albumin from the body (due to hemorrhage, excessive renal excretion, burn exudates, exudative enteropathy, or exfoliative dermatoses) and/or redistribution of albumin within the body (due to major surgery, cirrhosis with ascites, or various inflammatory conditions); prior to or during cardiopulmonary bypass surgery; and for the treatment of burns or cirrhosis.
A number of different preparations containing albumin for therapeutic use are or have been available commercially, including, for example, Albuminar® (Centeon/Aventis Behring), Buminate® (Baxter Laboratories), Plasbumin® (Bayer Biological), Albutein® (Alpha Therapeutic), Albumin (Human) (Immuno-U.S.), Albumarc® (American Red Cross) and Human Serum Albumin (Swiss Red Cross). Various plasma protein fraction products also are or have been available commercially, including, for example, Plasma-Plex® (Centeon/Aventis Behring), Protenate® (Baxter Laboratories), Plasmanate® (Bayer Biological) and Plasmatein® (Alpha Therapeutic).
Albumin is also used as a stabilizer in preparations of proteins, natural or recombinant, intended for therapeutic use. For example, albumin is presently found as a stabilizer in therapeutic preparations containing Factor VIII for hemophilia A (such as Recombinate® from Baxter-Hyland/Immuno), interferon beta-1b for multiple sclerosis (such as Betaseron® from Berlex Laboratories), erythropoietin for anemia (such as Epogen® from Amgen), alglucerase, a modified form of enzyme, β-glucocerebrosidase, for Gaucher's disease (such as Ceredase® from Genzyme) and antithrombin III for hereditary deficiency (such as Thrombate III® from Bayer or Atnativ® from Pharmacia & Upjohn). In such preparations, albumin may make up more than 99% of the total protein content.
Albumin is also found as a stabilizer in vaccine preparations, such as Tick-Born Encephalitis (TBE) Virus Vaccine and Measles, Mumps and Rubella Virus Vaccine Live (such as MMRII® from Merck), Rabies Vaccine (such as RabAvert® from Chiron and Imovax® from Pasteur-Merieux) and Oral Polio Vaccine (such as Evans Polio Vaccine® from Evans/Medeva). In such preparations, albumin may again make up more than 99% of the total protein content.
Preparations containing albumin are also used as nutrient formulations in media for cell culture, including the culture of cells (recombinant or otherwise) producing desired products, and vaccine production. Such preparations are available commercially from, for example, Sigma-Aldrich, Irvine Scientific, Intergen and Valley Biomedical.
Many preparations containing albumin that are prepared for human, veterinary, diagnostic and/or experimental use may contain unwanted and potentially dangerous biological contaminants or pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, prions or similar agents responsible, alone or in combination, for TSEs and/or single or multicellular parasites. Consequently, it is of utmost importance that any biological contaminant or pathogen in the biological material be inactivated before the product is used. This is especially critical when the material is to be administered directly to a patient, for example in blood transfusions, blood factor replacement therapy, organ transplants and other forms of human therapy corrected or treated by intravenous, intramuscular or other forms of injection or introduction. This is also critical for the various biological materials that are prepared in media or via culture of cells or recombinant cells which contain various types of plasma and/or plasma derivatives or other biologic materials and which may be subject to mycoplasma, prion, bacterial, viral and other biological contaminants or pathogens.
Most procedures for producing biological materials have involved methods that screen or test the biological materials for one or more particular biological contaminants or pathogens rather than removal or inactivation of the contaminant(s) or pathogen(s) from the material. Materials that test positive for a biological contaminant or pathogen are merely not used. Examples of screening procedures include the testing for a particular virus in human blood from blood donors. Such procedures, however, are not always reliable and are not able to detect the presence of certain viruses, particularly in very low numbers. This reduces the value or certainty of the test in view of the consequences associated with a false negative result. False negative results can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS). Furthermore, in some instances it can take weeks, if not months, to determine whether or not the material is contaminated. Therefore, it would be desirable to apply techniques that would kill or inactivate contaminants or pathogens during and/or after manufacturing the biological material.
Moreover, to date, there is no reliable test or assay for identifying prions within a biological material that is suitable for screening out potential donors or infected material. This serves to heighten the need for an effective means of destroying priors within a biological material, while still retaining the desired activity of that material.
In conducting experiments to determine the ability of technologies to inactivate viruses, the actual viruses of concern are seldom utilized. This is a result of safety concerns for the workers conducting the tests, and the difficulty and expense associated with the containment facilities and waste disposal. In their place, model viruses of the same family and class are used.
In general, it is acknowledged that the most difficult viruses to inactivate are those with an outer shell made up of proteins, and that among these, the most difficult to inactivate are those of the smallest size. This has been shown to be true for gamma irradiation and most other forms of radiation as these viruses' diminutive size is associated with a small genome. The magnitude of direct effects of radiation upon a molecule are directly proportional to the size of the molecule, that is the larger the target molecule, the greater the effect. As a corollary, it has been shown for gamma-irradiation that the smaller the viral genome, the higher the radiation dose required to inactive it.
Among the viruses of concern for both human and animal-derived biological materials, the smallest, and thus most difficult to inactivate, belong to the family of Parvoviruses and the slightly larger protein-coated Hepatitis virus. In humans, the Parvovirus B19, and Hepatitis A are the agents of concern. In porcine-derived materials, the smallest corresponding virus is Porcine Parvovirus. Since this virus is harmless to humans, it is frequently chosen as a model virus for the human B19 Parvovirus. The demonstration of inactivation of this model parvovirus is considered adequate proof that the method employed will kill human B19 virus and Hepatitis A, and by extension, that it will also kill the larger and less hardy viruses such as HIV, CMV, Hepatitis B and C and others.
More recent efforts have focussed on methods to remove or inactivate contaminants in the products. Such methods include heat treating, filtration and the addition of chemical inactivants or sensitizers to the product.
Current standards of the U.S. Food and Drug Administration require that heat treatment of preparations containing albumin be heated to approximately 60° C. for a minimum of 10 hours, which can be damaging to sensitive biological materials. Indeed, heat inactivation can destroy 50% or more of the biological activity of certain biological materials.
Filtration involves filtering the product in order to physically remove contaminants. Unfortunately, this method may also remove products that have a high molecular weight. Further, in certain cases, small viruses may not be removed by the filter.
The procedure of chemical sensitization involves the addition of noxious agents which bind to the DNA/RNA of the virus and which are activated either by UV or other radiation. This radiation produces reactive intermediates and/or free radicals which bind to the DNA/RNA of the virus, break the chemical bonds in the backbone of the DNA/RNA, and/or cross-link or complex it in such a way that the virus can no longer replicate. This procedure requires that unbound sensitizer is washed from products since the sensitizers are toxic, if not mutagenic or carcinogenic, and cannot be administered to a patient.
Irradiating a product with gamma radiation is another method of sterilizing a product. Gamma radiation is effective in destroying viruses and bacteria when given in high total doses (Keathly et al., “Is There Life After Irradiation? Part 2,” BioPharm July–August, 1993, and Leitman, Use of Blood Cell Irradiation in the Prevention of Post Transfusion Graft-vs-Host Disease,” Transfusion Science 10:219–239 (1989)). The published literature in this area, however, teaches that gamma radiation can be damaging to radiation sensitive products, such as blood, blood products, protein and protein-containing products. In particular, it has been shown that high radiation doses are injurious to red cells, platelets and granulocytes (Leitman). U.S. Pat. No. 4,620,908 discloses that protein products must be frozen prior to irradiation in order to maintain the viability of the protein product. This patent concludes that “[i]f the gamma irradiation were applied while the protein material was at, for example, ambient temperature, the material would be also completely destroyed, that is the activity of the material would be rendered so low as to be virtually ineffective”. Unfortunately, many sensitive biological materials, such as monoclonal antibodies (Mab), may lose viability and activity if subjected to freezing for irradiation purposes and then thawing prior to administration to a patient.
In view of the difficulties discussed above, there remains a need for methods of sterilizing preparations containing albumin that are effective for reducing the level of active biological contaminants or pathogens without an adverse effect on the preparation.